Inactivation Of SERCA2 Cysteine 674 Increases Blood Pressure By Induction Of Endoplasmic Reticulum Stress And Soluble Epoxide Hydrolase

Hypertension is a common multifactorial disease and is an important risk factor for cardiovascular and cerebrovascular diseases such as stroke,heart failure and kidney failure.It affects approximately 1.4 billion people worldwide in which about 9 million people die from complications of hypertension,so hypertension is a major public health problem.For several decades researchers all over the world have carried out extensive and in-depth studies on hypertension and made a lot of significant advances.The pathogenesis of hypertension is so complicated that the underlying mechanisms have not been fully elucidated.However,several hypotheses of hypertensive pathogenesis have been identified including sympathetic hyperactivity,vascular dysfunction,sodium retention and so on.Therefore,it is of great significance to clarify the regulation mechanism of blood pressure（BP）for the treatment of hypertension.Sarcoplasmic/endoplasmic reticulum calcium ATPase 2（SERCA2）is the most widely distributed SERCA subtype and is the key enzyme to maintain calcium homeostasis by taking up calcium from intracellular space to sarcoplasmic reticulum（SR）and endoplasmic reticulum（ER）.The S-glutathiolation of thiol at cysteine 674（C674）is key to increase the activity of SERCA2 under physiological conditions,but this post-translational protein modification is prevented by the irreversible oxidation of C674 thiol in pathological situations accompanied with high levels of reactive oxide species（ROS）,such as aging,diabetes mellitus,and atherosclerosis,which are all risk factors of hypertension.Angiotensin II（Ang II）is a common inducer to hypertension.We found that the irreversible oxidation of C674 in the kidney of Ang II-induced hypertensive mice was significantly increased,suggesting the inactivation of C674 is involved in the occurrence and development of high blood pressure.To our knowledge there are no papers reporting the role of C674 in regulation of high blood pressure,so this thesis delves into whether the irreversible oxidation of SERCA2 C674 increases blood pressure and has a preliminary discussion on pharmaceutical intervention based on these molecular mechanisms for the treatment of hypertension.Heterozygous SERCA2 C674S knock-in（SKI）mice in C57BL/6J background were constructed to represent C674 inactivation induced by the partial irreversible oxidation of C674 under hypertension-prone conditions,aiming to explore the relationship between the inactivation of C674 and hypertension.Compared with wild type（WT）mice,SKI mice had higher BP,lower urine volume and sodium excretion,suggesting C674 inactivation induces the water and sodium retention and thus elevates blood pressure.Na⁺/K⁺-ATPase is the key enzyme in regulating water and sodium reabsorption in renal proximal tubules（RPT）,and its increased activity will cause water and sodium retention.Histological results showed that there was no difference in the renal histological structure between WT mice and SKI mice.We found that the activity of Na⁺/K⁺-ATPase was increased in SKI RPT cells compared with WT RPT cells.These results suggest that high blood pressure induced by C647 inactivation is attributed to renal functional alteration instead of pathological changes in renal structure.In order to explore whether C674 inactivation affects SERCA2 function in the kidney,we examined the expression of SERCA2 in the renal cortex and RPT cells respectively in WT mice and SKI mice.The results showed that there was no difference in the m RNA abundance of both SERCA2a and SERCA2b in renal cortex between WT mice and SKI mice,although the relative copy number of SERCA2b was about 100 times higher than that of SERCA2a,indicating that SERCA2b is the predominant one among all SERCA isoforms in the kidney.There was no difference in protein expression levels of SERCA2between the two groups.In addition,the intracellular calcium levels of SKI RPT cells was higher than that of WT RPT cells,indicating that C674 inactivation inhibits the normal function of SERCA2 in RPT cells without affecting the expression of SERCA2.SERCA2 is the only enzyme in the ER to take up intracellular calcium into ER to maintain ER calcium stores,which is imperative for ER normal function,and the sustained ER calcium depletion causes ER stress.In order to identify if ER stress occurs in the kidney of SKI mice,we examined ER stress markers in renal cortex and RPT cells in WT mice and SKI mice.We found that the inactivation of C674 significantly up-regulated the protein expression of ER stress markers phospho-protein kinase R-like ER kinase（p-PERK）,immunoglobulin binding protein（BIP）,activating transcription factor 6（ATF6）and C/EBP homologous protein（CHOP）.Moreover,the administration of ER stress inhibitor 4-phenylbutyrate（4-PBA）reversed the elevated BP,decreased urine volume and urinary sodium excretion in SKI mice similar to the ones in WT mice without affecting by 4-PBA,which was associated with the decreased Na⁺/K⁺-ATPase in SKI RPT by 4-PBA treatment.In order to study the causes of sodium retention in SKI mice,we screened genes to determine which factors contributed to the reduction of water and sodium excretion and elevated BP in SKI mice,and results showed that both soluble epoxide hydrolase（s EH）and dopamine D1 receptor（D1R）were affected.Compared with WT mice,the s EH of SKI mice increased and D1R decreased at the level of m RNA,which were further supported at the level of protein in both renal cortex and primary RPT cells.s EH metabolizes epoxyeicosatrienoic acid（EETs）to less active dihydroxyeicosatrienoic acids（DHETs）,thus inhibits sodium excretion by enhancing the activity of Na⁺/K⁺-ATPase in RPT.On the contrary,activation of D1R promotes sodium excretion by inhibiting Na⁺/K⁺-ATPase in RPT.Therefore,the increase of s EH and the decrease of D1R by C674 inactivation could reduce sodium excretion by enhancing the activity of Na⁺/K⁺-ATPase,thus causing the retention of water and sodium,and ultimately increasing BP.Next,we examined whether persistent ER stress induced by C674 inactivation explained the increase in s EH expression and the decrease in D1R expression.In WT RPT cells,4-PBA partially down-regulated ER stress markers,while s EH and D1R were not affected by 4-PBA.However,4-PBA down-regulated ER stress markers as well as s EH expression,and up-regulated the reduced D1R expression in SKI RPT cells.These results indicate that the persistent ER stress in the renal cortex of SKI mice controls sodium excretion and BP by regulating s EH and D1R.To explore the role of s EH in elevated BP of SKI mice,we applied s EH inhibitor 1-trifluoromethoxyphenyl-3-（1-propionylpiperidin-4-yl）urea（TPPU）to inhibit s EH activity in RPT cells from both WT mice and SKI mice.In WT RPT cells,TPPU itself did not affect the expression of s EH compared with solvent control,nor did it affect the expression of ER stress markers and D1R.Although TPPU did not affect s EH expression,it did reverse the increased ER stress markers and decreased D1R expression in SKI RPT cells.These results suggest that ER stress and s EH might regulate each other,and both in the upstream of D1R.Similar to 4-PBA,TPPU in vivo reversed the elevated BP and the decreased daily urine volume and urinary sodium excretion in SKI mice.The increased Na⁺/K⁺-ATPase activity in SKI RPT cells was also reversed by TPPU,indicating upregulation of s EH is a major cause of elevated BP in SKI mice.To investigate the roles of the up-regulated s EH in SKI mice,we made s EH over-expressed in rat RPT cells.The results showed that overexpression of s EH increased the expression of ER stress markers and decreased the expression of D1R,confirming that ER stress and s EH were mutually regulated,and both in the upstream of D1R.Although s EH and ER stress can regulate each other,we infer that ER stress inducement precedes upregulation of s EH in SKI mice based on the basic role of SERCA2 in maintaining intracellular calcium homeostasis and ER calcium homeostasis.We made SERCA2b C674 and SERCA2b S674 over-expressed in rat RPT cells to further confirm that C674 inactivation directly regulates ER stress,s EH and D1R.The treatment of hypertension is a hot medical issue in the world.Based on the study of hypertension,there are several first-line anti-hypertensive drugs in the market,including diuretics,βreceptor blockers,angiotensin-converting enzyme inhibitors,calcium antagonists and Ang II type 1 receptor blockers.With an increase in the prevalence of hypertension and lower rates of hypertension control,it is urgent for researchers to further explore the pathogenesis of hypertension and to develop new drugs.Thereby,SKI mice were used as a novel hypertensive animal model to study the role of SERCA activator CDN1163 and[6]-Gingerol in the treatment of hypertension.We applied CDN1163 and[6]-Gingerol in SKI RPT cells and found that the two SERCA activators decreased intracellular Ca²⁺levels,ER stress markers and s EH protein expression levels,increased D1R protein expression levels,and inhibited Na⁺/K⁺-ATPase activity.Furthermore,CDN1163 and[6]-Gingerol in vivo therapy reversed the elevated BP,the decreased daily urine volume and urine sodium excretion in SKI mice.In summary,this study has provided solid evidence for the first time that SERCA2is involved in maintaining BP and that C674 redox status plays an important part in BP control.Under pathological conditions,the inactivation of SERCA2 C674 elevates BP by inducing water and sodium retention.One possible mechanism is that renal C674inactivation induces ER stress and s EH,and inhibits the expression of D1R to enhance the activity of Na⁺/K⁺-ATPase in RPT cells,which leads to water and sodium retention and elevates BP.We use SKI mice as a hypertensive model and has a preliminary discussion on the effect of 4-PBA,TPPU,CDN1163 and[6]-Gingerol in the treatment of hypertension.Pharmacological inhibition of s EH activity or ER stress,or activation of SERCA2,may exert therapeutic benefits in the treatment of hypertension under these conditions.